Use of inhibitors for the treatment of rtk-hyperfunction-induced disorders, particularly cancer

ABSTRACT

The present invention concerns the use of inhibitors for the treatment and/or prophylaxis of diseases which are the consequence of increased receptor tyrosine kinase activity, particularly cancer. The use is particularly directed towards inhibition or lowering of the overexpression and/or altered activity of receptor tyrosine kinases (RTKs). In particular, this altered activity of receptor tyrosine kinase can be triggered by a mutation of FGFR-4, wherein this mutation is in particular a point mutation in the transmembrane domain of FGFR-4 and leads to an exchange of a hydrophobic amino acid for a hydrophilic amino acid. The invention further concerns the use of an inhibitor directed against FGFR-4, for the treatment and/or prophylaxis of cancer. Furthermore, the invention concerns a mutated FGFR-4, which leads to over-expression and/or altered activity in cells. Finally, the invention concerns a DNA and RNA sequence of a mutated FGFR-4 molecule. Finally, in addition the invention concerns a pharmaceutical composition, containing the inhibitor as described above and further a diagnostic and screening procedure.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a divisional of copending U.S. patentapplication Ser. No. 10/649,413, filed Aug. 27, 2003, which is adivisional of U.S. patent application Ser. No. 09/600,826 filed Sep. 7,2000, issued as U.S. Pat. No. 6,770,742, all of which are herebyincorporated by reference.

INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY FILED

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 9,599 Byte ASCII (Text) file named“705943SequenceListing.TXT,” created on Jan. 18, 2010.

The present invention concerns the use of inhibitors for the treatmentand/or prophylaxis of diseases which are the consequence of increasedreceptor tyrosine kinase activity, particularly cancer. The use isparticularly directed towards inhibition or lowering of theoverexpression and/or altered activity of receptor tyrosine kinases(RTKs). In particular, this altered activity of receptor tyrosine kinasecan be triggered by a mutation of FGFR-4, wherein this mutation is inparticular a point mutation in the transmembrane domain of FGFR-4 andleads to the exchange of a hydrophobic amino acid for a hydrophilicamino acid. The invention further concerns the use of the inhibitors ofFGFR kinases, particularly for the treatment and/or prophylaxis ofcancer. Furthermore, the invention concerns a mutated FGFR-4, whichleads to overexpression and/or altered activity in cells. Finally, theinvention concerns a DNA and RNA sequence of a mutated FGFR-4 molecule.Finally, in addition the invention concerns a pharmaceuticalcomposition, containing the inhibitor as described above and, further, adiagnostic and screening procedure.

Cell growth is a carefully regulated process dependent on the specificneeds of an organism. In a young organism, the cell division rateexceeds the cell death rate, which leads to an increase in the size ofthe organism. In an adult organism, the new formation of cells and celldeath are balanced so that a “steady state” arises. In rare cases,however, the control of cell multiplication breaks down and the cellsbegin to grow and to divide, although no specific need for a highernumber of cells of this type exists in the organism. This uncontrolledcell growth is the cause of cancer. Factors that can provoke theuncontrolled cell growth, some-times associated with metastasisformation, are often of a chemical nature, but can also be of a physicalnature, such as for example radioactive radiation. Another cause of thetriggering of cancer are genetic peculiarities or mutations in a certainorganism, which sooner or later lead to the cells degenerating.

Up to now, it has still not been possible satisfactorily to elucidatethe processes which control normal growth and differentiation, forexample in the breast. In addition to hormonal control, there is also acomplex network of different, locally generated growth factors whichintervene in the development of the mammary cells. The precise causes ofthe occurrence of cancer in mammary cells are as unclear and unknown asthey are diverse, as is also the case with other cells. Alterations inoncogenes and tumour suppressor genes appear to play an important partin breast cancer carcinogenesis. In addition, reinforced stimulation byregulatory factors which arise in genetically altered cells can lead toincreased progression of cell growth.

At present, essentially two alternatives are available for the treatmentof cancer. Either the cancer cells are successfully removed from thediseased organism completely by a surgical intervention, or attempts aremade to render the degenerated cells in the organism harmless, forexample by administration of medicaments (chemotherapy) or by physicaltherapeutic procedures, such as irradiation.

In chemotherapy, medicaments are often used which in some form intervenein the DNA metabolism and damage rapidly growing cells, which have toproduce higher DNA metabolic capacity, more strongly than cells whichare dividing slowly or not at all. However, a severe disadvantage ofmany chemotherapeutic drugs is the low specificity of the activesubstance used, as a result of which healthy cells are also damagedduring the chemotherapy. This low specificity of the active substancesfurther requires that their dosage must in each case be such that as fewas possible healthy cells are damaged, with simultaneous killing of thecancer cells. This is often not possible, and the cancer patient diesbecause of the ever further spreading cancer cells, which in the finalstages cause the failure of vital functions.

It is assumed that the overexpression and/or altered activity of certaingrowth factor receptors contribute to the intensified growth of manyneoplasms, including breast cancer. For example, the overexpression ofEGFR, i.e. epidermal factor receptor, or ERB B-2 receptor in breasttumours has been linked with a poor prognosis. FGF (historically:fibroblast growth factor) proteins could also be involved in thedevelopment of cancer in breast glands or of other cancer; however theresults in this regard are contradictory or are inconclusive.

The FGFs constitute a large family of peptide regulatory factors, ofwhich 9 members are so far known. Eight of these have been wellcharacterised in man (Basilico and Moscatelli, 1992; Coulier et al.,1993). The FGFs operate via high-affinity tyrosine kinase receptors,which are coded for by at least four different genes. Further, the FGFsare multifunctional, regulatory peptides which could have an effect notonly on tumorigenesis but could also play a major part in cardiovasculardiseases, reconstruction after tissue injury, neurobiology and embryonicdevelopment. The acidic and basic FGFs (aFGF and bFGF) were the firstand are the best characterised members of the family. In vivo it couldfor example be shown that FGFs are involved in mesodermal induction inembryogenesis (Slack et al., 1987; Kimelman et al., 1988), and alsoinvolvement in angiogenesis (Thomas et al., 1985; Thompson et al., 1989;Folkmann and Klagsbrun, 1987).

For the corresponding receptors (FGFRs), four similar genes coding forthem have been identified. These genes code for structurally relatedproteins with an extracellular domain which consists of threeimmunoglobulin loops and an acid portion, a hydrophobic trans-membranedomain and an intracellular domain, which incorporates a tyrosine kinaseactivity. For two of these genes, FGFR-1 and FGFR-2, it could be shownthat they have multiple transcripts, which arise by alternative splicing(Givol and Yayon, 1992 and Johnson and Williams, 1993). Splice variantswhich arise from these genes differ with respect to the number ofimmunoglobulin-like domains in the extracellular region of the receptorand in the sequence for the second half of the third immunoglobulindomain, which can arise from alternative exons. In addition,transmembrane and juxtamembrane shortenings or deletions can arise,which can generate secreted or kinase-inactive protein products.

For FGFR-3, it was possible to find alternative transcripts andcorresponding isoforms, but for FGFR-4 there is only a single knownprotein product. Because of the large number of FGFR genes andtranscripts and the lack in many protein products of a specificity fordefined FGFs, it is difficult to determine the action of a specificligand on a specific receptor. Hence, correlations between specific FGFreceptors and defined diseases can only be established with greatdifficulty, let alone a correlation of a particular mechanism of actionof a defined receptor with a disease. Accordingly, it is difficulteffectively to treat diseases, especially the complex disease picturecancer, utilising the FGFRs.

Hence it is an object of the present invention to specify a possibletreatment and/or prophylaxis of somatic disorders, in the development ofwhich receptor tyrosine kinases (RTKs) are involved, particularlycancer. In particular, it is an object of the present invention toinhibit and/or to lower overexpression and/or altered, for exampleconstitutive activity of receptor tyrosine kinases.

It is further an object of the present invention to inhibit and/or tolower the altered activity of the receptor tyrosine kinase of a mutatedFGFR-4.

It is a further an object of the present invention to specify a furtherRTK which is involved in carcinogenesis and/or metastasis formation.Further, it is an object of the present invention to specify a DNAsequence or corresponding RNA sequence of the RTK.

It is a further an object of the present invention to specify improveddiagnostic or differential diagnostic and screening procedures.

Finally it is an object of the present invention to specify apharmaceutical composition, with which in particular cancer can betreated.

These objects are achieved by the objects of the independent claims. Thedependent claims specify preferred developments of the invention.

For the better understanding of the present invention, the terms usedherein are explained in more detail.

By “inhibitor” is understood any substance which inhibits the RTK orlowers their activity. This can be a low-molecular weight substancedirected against the RTK, a kinase-inactive receptor or an anti-receptorantibody.

By “kinase-inactive receptor” is understood any, receptor which nolonger has any tyrosine kinase activity.

By “receptor tyrosine kinase” [sic] is understood any receptor which hastyrosine kinase activity. The expression includes growth factorreceptors which have tyrosine kinase activity, and also HER2 or themet-receptors.

By “RTK-Hyperfunction” is understood overexpression (see below) and/oraltered activity (see below).

“Defective signal transfer activities” means that a mutated receptor isno longer capable of converting an extracellular growth signal oranother signal into an intracellular signal, in the sense that thisdefective signal generation no longer depends on the presence of aligand, for example the growth factor.

“Growth factor” means any mitogenic chemical, usually a polypeptide,which inter alia is secreted by normal and/or transformed mammaliancells, and which plays a significant part in the regulation of cellgrowth, in particular in the stimulation of the proliferation of thecells and the maintenance of their viability. The term “growth factor”for example includes epidermal growth factor (EGF), platelet-derivedgrowth factor (PDGF) and nerve growth factor (NGF), and also FGF, namelyfibroblast growth factor.

By “mutated receptor tyrosine kinase” is understood a receptor tyrosinekinase which by comparison with the wild type receptor contains astructural alteration, so that the receptor has a different, e.g. nolonger regulable, tyrosine kinase activity from the wild type receptor.One class of mutations leads to altered activity of the RTK.

By “wild type growth factor receptor” or “wild type” receptor isunderstood a naturally occurring growth factor receptor or receptor thatbears the non-mutated amino acid sequence. The “wild type” correspondsto the receptor variant most commonly occurring in the population.

By “extracellular domain” of the growth factor receptor or receptor isunderstood the part of the receptor which normally projects out of thecell into the extracellular surroundings. The extracellular domain forexample includes the part of the receptor to which a growth factor oranother molecule (ligand) binds.

By “transmembrane region” of the growth factor receptor or receptor isunderstood the hydrophobic portion of the receptor, which is normallylocated in the cell membrane of the cell which expresses the receptor.

By “tyrosine kinase domain” or “cytoplasmic domain” of the growth factorreceptor or receptor is understood the portion of the receptor which isnormally situated inside the cell, and brings about thetransphosphorylation of tyrosine residues.

By “an effective quantity” is understood a quantity of the compositionaccording to the invention which can achieve the desired therapeuticeffect.

By “fibroblast growth factor (FGF)” is understood a mitogenicpolypeptide which influences the growth and other properties of cells,inter alia of fibroblasts.

By “overexpression” is understood increased production of RTK protein bya cell as compared to the wild type. This can for example be triggeredby gene amplification of the RTK gene and lead to excessive,uncontrolled cell division activity.

By “altered activity” is understood permanent activity of a signaltransfer route mediated by growth factor receptors. Thus with an alteredRTK the kinase activity is also present when no ligand is present.

According to the present invention, it could be shown that a mutatedFGFR-4 can lead to overexpression and/or altered activity of thecorresponding receptor tyrosine kinase in cells and hence lead tocancer.

Growth factor receptors play a decisive part in the development andmultiplication of human cancer cells. In healthy cells, the growthfactor receptors are inter alia involved in the control of cell growth,but also in differentiation, cell migration, etc. The actual signal forthe cell division is the growth factor, which is formed depending on theneeds of the organism. The receptor undertakes the function of signaltransfer, i.e. it is involved in the conversion of the extracellulargrowth signal into cell division activity in the inside of the cell.With many growth factor receptors, their ability, after binding of thegrowth factor to the extracellular domain, to transfer phosphateresidues onto tyrosine residues in proteins plays a decisive part. Thesereceptors are also described as receptor tyrosine kinases. A review ofreceptor tyrosine kinases is to be found in Yarden Y and Ullrich A, Rev.Biochem. 1988, 57, 443-78. The dimerisation of these growth factorreceptors after binding of the growth factor is a further importantevent in the process of signal transfer. The conversion of anextracellular signal into an intracellular signal mediated by growthfactor receptors with tyrosine kinase activity can be broken down intothe following five steps:

1. The binding of the growth factor (also described as ligand) to theextracellular domain of the receptor induces a conformational change;this causes2. dimerisation of receptors with altered conformation; with3. simultaneous induction of kinase activity;4. transphosphorylation of tyrosine residues in the receptor dimer,which once again creates and stabilises an activated receptorconformation; and5. phosphorylation of polypeptide substrates and interaction withcellular factors.

Uncontrolled hyperfunction of this signal transfer chain for examplebecause of the over-expression or altered activity of the receptor caninter alia lead to increased division activity of the relevant cells andin the extreme case to a degenerated cancer cell. A review concerninggrowth factor receptors and their function in signal transfer from theextracellular to the intracellular milieu, and the possible influence ofabnormally expressed receptors on carcinogenesis, is given in Ullrich Aand Schlessinger J (1990) Cell 61, 203-212.

It has now surprisingly been found that in the five-stage signaltransfer chain explained above, a mutated FGFR-4 results in increasedsignal transfer activity, in the development of which the alteredactivity of mutated RTK is decisively involved.

Hence according to claim 1 of the present invention at least oneinhibitor of a receptor tyrosine kinase is used for the treatment and/orprophylaxis of RTK-hyperfunction-induced disorders, particularly cancer.Furthermore, according to the invention diseases or somatic disorderswhich are the consequence of a hyperproliferation of tissues and/orincreased invasivity of tissues attributable to increased signaltransfer can also be eliminated or alleviated.

As inhibitor, as well as low-molecular weight substances, for example atleast one kinase-inactive receptor can be used. Through the use of theinhibitor, e.g. of the kinase-inactive receptor, the altered activity ofthe receptor tyrosine kinase can be inhibited and/or lowered. As hasalready previously been stated, the overexpression and/or alteredactivity of growth factor receptors is an important factor in thetriggering or the progression of cancer. The overexpression of EGFR orthe Erb B-2 receptor in breast tumours has for example been associatedwith a poor prognosis (see above). Hence inhibition of thisoverexpression and/or altered activity is an important component in thetreatment and/or prophylaxis of cancer. FGFR-4 is tissue-specificallyswitched off during embryogenesis. However, it is present in 30% ofbreast cancer patients; it is not detectable in the tissue of healthysubjects. The use of inhibitors for receptor tyrosine kinase leads to alowering or complete inhibition of the over-expression and/or alteredactivity. Likewise, the use of kinase-inactive receptors leads to alowering and/or complete inhibition of the activity of the receptortyrosine kinases, since the kinase function of the heterodimer is nolonger capable of signal transfer. The action of kinase-inactivereceptors is based on the fact that non-functional heterodimers areformed (dilution effect). A lack of signal transfer leads to preventionof the transmission of the overexpressed and/or altered active signal,as a result of which the signal is prevented from conversion into abiological response of the cell. As a result, through this inhibition ofthe receptor tyrosine kinase or through these kinase-inactive receptors,it is possible effectively and positively to intervene in the treatmentand/or prophylaxis of cancer.

It has surprisingly been found that the FGFR-4 mutation also occurs inthe germ line of healthy persons. It is assumed that the germ linemutation leads to a genetic predisposition, which renders the personsconcerned susceptible to the outbreak of various diseases. In connectionwith carcinogenesis, it is assumed that the increase expression of themutated receptor in the tumour tissue is involved in the carcinogenesis.The germ line mutation is further regarded as a predisposition interalia for the following diseases: arteriosclerosis, leukaemia, lymphoma,hepatic cell carcinoma and cholangiocarcinoma.

Consequently, the present invention makes a further genetic markeravailable, which is found to be extremely helpful in the diagnosis andearly recognition of various diseases and susceptibility to these.

The present invention therefore also concerns a procedure for thedetection of a nucleic acid which codes for FGFR-4 in case material,whereby in particular mutations of the receptor-coding nucleic acid aredetected. This can for example be effected by hybridisation witholigonucleotide probes, which can specifically indicate the presence orabsence of a mutation, in particular a point mutation. In this, forexample a “mismatch” between mutated nucleic acid and oligonucleotide isutilised such that if a “mismatch” is present a hybridisation does nottake place and hence there is no signal. Alternatively, mutations canalso be detected by amplification of the nucleic acid with specificFGFR-4 PCR primers and subsequent cleavage with suitable restrictionendonucleases. If for example a mutation affects the recognitionsequence of a restriction endonuclease, such that for example themutated recognition sequence is no longer recognised as a cleavage siteby the restriction endonuclease, this leads to a different restrictionfragment than in the non-mutated wild type. By means of the PCR,restriction fragments can be specifically detected, so that in thestated case for example a larger restriction fragment is present in themutant compared to the wild type. Alternatively, however, a mutation canalso lead to the creation of a new restriction cleavage site, as aresult of which a “wild-type fragment” after cleavage with theappropriate enzyme becomes smaller in the mutant. The mutation in thetransmembrane domain of FGFR-4, at position 388 of the sequence, asdeposited in the EMBL Gene Bank/DDBJ under X57205, which leads to anexchange of Gly in the wild type for Arg in the mutant, concerns therecognition sequence GGWCC of the restriction endonuclease BstN1. As aresult, two new restriction fragments of 80 and 29 b.p. are formed,which can inter alia be detected by restriction analysis.

According to the present invention, it could further be shown thatoverexpression and in particular altered activity of the RTK leads toincreased invasivity, i.e. to intensified metastasis formation. Sincemetastasis formation is one of the main problems of cancer, this meansthat the inhibition or lowering of the overexpression and/or alteredactivity will lead to an effective agent in the combating of cancer, bywhich in particular metastasis formation is inhibited.

Possible inhibitors are for example described in Mohammadi et al.(1997).

Preferably according to the invention an intervention is made into anoverexpression and/or altered activity of the receptor tyrosine kinase,which is triggered by a mutation of FGFR-4. This mutation can be one orseveral point mutations. In particular, the mutation/mutations occur inthe transmembrane domain of FGFR-4, as a result of which in particular ahydro-phobic amino acid is exchanged for a hydrophilic amino acid.

It is already known that point mutations which have led to an exchangeof hydrophobic for hydrophilic amino acids in FGFR-3 are associated withcertain diseases. Thus for example, an altered activity of fibroblastgrowth factor receptor 3 due to a point mutation in the transmembranedomain has been found in achondroplasia. Achondroplasia, which is themost commonly occurring genetic form of dwarfism, is an autosomaldominant disorder, which is essentially based on a defect in thematuration process of certain bones. It could be shown thatachondroplasia is triggered by a Gly to Arg substitution in thetransmembrane domain of FGFR-3. It could further be shown that that theArg mutation in FGFR-3 activates the kinase function of the dimericreceptor. The Arg point mutation also leads to a ligand-independentstimulation of the tyrosine kinase activity of FGFR-3 itself and tostrongly increased altered levels of phosphotyrosine on the receptor.These results suggest that the molecular basis of achondroplasia isunregulated signal transfer by FGFR-3.

A further mutation in the transmembrane domain of FGFR-3 is an exchangealanine for glutamine. This amino acid exchange leads to anotherdisease, namely to Crouzon's disease with acanthosis nigricans.

According to the present invention, it was established that mutations inFGFR-4, especially point mutations in the transmembrane domain, whichlead to an exchange of a hydrophobic for a hydrophilic amino acid, areinvolved in the triggering and poor prognosis for cancer, on account ofwhich inhibition of receptor tyrosine kinases or the use ofkinase-inactive receptors are suitable for the treatment and/orprophylaxis of cancer, wherein the receptor tyrosine kinases areoverexpressed or active in an altered way owing to a mutation.

In particular, for the point mutation at position 388, which leads to anexchange of glycine for arginine, it could be shown that as a result ofthis the receptor tyrosine kinases become active in an altered way, andthis homo- and heterozygotically results in signal transfer withoutligand stimulation, as a result of which in turn an uncontrollablegrowth of cells can be triggered. In the worst case, this uncontrolledgrowth leads to cancer. The transmembrane domain then has the sequence(ID No. 1):

RYTDIILYASGSLALAVLLLLARLY,while the non-mutated domain has the following sequence (ID No. 2):

RYTDIILYASGSLALAVLLLLAGLY.

Without being bound to one theory, it is assumed that the activation ofthe receptor tyrosine kinase which bears one of the aforesaid pointmutations and in particular the point mutation at position 388, whichleads to an exchange of glycine for arginine, is based on astabilisation of the receptor in a dimeric conformation, which occursbecause of interactions through which changes in the transmembranedomain were made possible. The intensified formation of aligand-independent dimer leads to increased receptor tyrosine kinaseactivity and cellular transformation. Other possibilities for the effectof the point mutations on the triggering of cancer may for example havea basis in that the mutation acts on the signal transfer by FGFR-4, inthat the receptor migration through the membrane is prevented, thereceptor dimerisation with itself or with other FGFRs is disturbed, orin that the tyrosine kinase activity of the receptor is affected.

According to the present invention, it could be shown that 56% ofpatients from St Petersburg with breast tumours (study of biopsies)carried the mutation at position 388, which is linked with an exchangeof glycine for arginine. Of these, 45% were heterozygotic and 11%homo-zygotic. This significantly high proportion suggests a link betweenthe point mutation at position 388 and the occurrence of breast cancer.

In a further study, in which German patients with breast tumours werestudied, only 43% of the patient showed the point mutations at position388. From the study with normal tissues of cancer patients and DNA fromthe tissue of normal individuals, it can be inferred that the mutationis a germ line mutation.

Furthermore, genomic DNA and cDNA from cell lines was also studied, inorder to determine the proportion of point mutations at position 388.The cell lines studied derived from breast tumours, normal breastepithelial cell lines as a comparison, squamous cell carcinoma,glioblastomas, neuroblastomas and uterine cancer. With all cell lines,except for the normal breast epithelial cell lines, a significantpercentage of the point mutation at position 388 in the FGFR-4 moleculecould be found. Hence the above-mentioned use of inhibitors orkinase-inactive receptors is especially suitable for the treatment ofcarcinomas. Here the treatment of neuroblastomas, uterine cancer andpancreatic cancer, but also other types of cancer, seems especiallypromising.

Particularly preferred is the use of inhibitors which inhibit a mutatedFGFR-4, especially with the mutation Gly→Arg at position 388 in thetransmembrane domain.

Further, the present invention concerns a mutated FGFR-4, which leads tooverexpression and/or altered activity of the receptor in cells.Preferably, this mutated FGFR-4 is characterised in that a hydrophobicamino acid in the wild type receptor has been exchanged for ahydrophilic amino acid in the mutated receptor. Especially preferred isa mutation which is a point mutation and occurs in the transmembranedomain. Still more preferably, the point mutation occurs at position388, as a result of which preferably a glycine is replaced by arginine.

Hitherto, it was assumed among experts that only one FGFR-4 occurs,which is not mutated. Mutated FGFR-4 was unknown. It was thereforesurprising that it could be shown according to the present inventionthat a mutated FGFR-4 exists. In particular, according to the presentinvention a connection between the mutations, in particular the pointmutation at position 388, and the occurrence of cancer could bedemonstrated. Furthermore, the germ line mutation in healthy persons hasbeen connected with the genetic predisposition for the occurrence interalia of arteriosclerosis.

The invention further concerns a DNA molecule containing a sequencewhich codes for a mutated FGFR-4. The invention also includes an RNAmolecule, containing an RNA sequence which codes for a mutated FGFR-4.The above sequences can be used for diagnosis of cancer. In this, thesequences can specifically recognise the mutations in the FGFR-4. Thepresence of the mutation in the FGFR-4 is linked with a poor prognosisfor the treatment of the cancer. The reason for this could be aggressivegrowth behaviour of the corresponding tumour.

Apart from this, the invention concerns a procedure for the differentialdiagnosis of breast cancer, wherein the patient's nucleic acid isbrought into contact with one of the DNAs and/or RNAs described above,so that a signal is obtained, which indicates the presence and/orabsence of mutated FGFR-4. Finally, the present invention concerns apharmaceutical composition, containing the inhibitor or thekinase-inactive receptor, as described above. Apart from this, theinvention concerns a screening procedure for the identification ofinhibitors of tyrosine kinase activity, wherein the receptor accordingto the invention is brought into contact with potential inhibitors andthe tyrosine kinase activity in the presence and/or absence of theinhibitor is determined.

Further, the detection of the presence of a mutation can also beperformed by PCR and subsequent restriction enzyme cleavage, as alreadydescribed in more detail above.

Finally, other molecular biological diagnostic procedures are also apossibility.

Further, the object of the invention is an antibody which specificallyreacts with a mutated FGFR-4 according to the invention. “Specific” inthe sense of the invention means that the antibody according to theinvention binds to the mutated, but not to the non-mutated, receptor.

Below, the invention is described in detail by the figures and examples.

Here:

FIG. 1 shows SDS PAGE of an immunoprecipitation of FGFR-4 (thephosphorylated FGF receptor-4 is marked by an arrow),

FIG. 2 a polyacrylamide gel of the mutated FGFR-4,

FIG. 3 a sequence analysis of the transmembrane domain of FGFR-4,

FIG. 4 the correlation between the FGFR-4 mutation G388R and the lymphnode metastasis formation status (n=number of patients, p=P value) and

FIG. 5 the correlation between the FGFR-4 mutation G388R and therelapse-free survival time (n=number of patients, p=P value).

EXAMPLES

Cell Culture. The human cell lines MDA-MB-453, ZR 75-1, K562 and SKBr3were obtained from the ATCC. The individual supply sources can be foundin the table at the end. MDA-MB-453, K562 and ZR 75-1 were cultivated inRPMI (Gibco, Eggenstein) containing 10% foetal calf serum (Sigma,Taufkirchen). SKBR3 was cultivated in McCoy's 5a (Gibco, Egenstein)containing 15% foetal calf serum. All cell culture media containedpenicillin/streptomycin (Sigma, Taufkirchen). The cells were incubatedat 37° C. in a water-vapour saturated atmosphere and 8% CO₂.

Cloning of FGFR-4^(388Arg)/wt. For preparation of RNA from K562 andMDA-MB-453 cells, 3×10⁷ cells were lysed with guanidinium isothiocyanateand purified by ultracentrifugation in a CsCl gradient. The cDNAsynthesis was effected with reverse transcriptase (Boehringer, Mannheim)and 10 μmol of “random oligonucleotides” in each case, according to themanufacturer's instructions. 0.5 μl were used in a subsequent PCRreaction.

FGFR-4^(388Arg) and FGFR-4 wt were amplified by the PCR reaction. Forthis, the following primers were used:sense-GCTCAGAGGGCGGGCGGGGGTGCCGGCCG [SEQ ID NO: 3]; anti-senseCCGCTCGAGTGCCTGCACAGCCTTGAGCCTTGC [SEQ ID NO: 4]. For the PCR reaction,the following were used: 1.5 U/25 μl Expand-Polymerase (Boehringer,Mannheim) and reaction buffer according to the manufacturer'sinstructions: 200 μM dNTP's; 0.01% v/v Triton X100; 10% v/v DMSO, and0.2 μmol each of sense and α-sense primer. The following reaction stepswere performed: 35 cycles, 94° C. 1 min, 64° C. 1 min, 72° C. 2.5 min.MDA-MB-453 cDNA was used for the cloning of FGFR-4^(388Arg), and K562cDNA for the cloning of FGFR-4 wt. The PCR products were cloned in thepcDNA3 vector (Invitrogen). In this way, both a FGFR-4 with the G388Rand also a wild type FGFR-4 could be obtained for further tests.

Amplification of the transmembrane domain of FGFR-4. The followingprimers were used: sense-GACCGCAGCAGCGCCCGAGGCCAG [SEQ ID NO: 5];anti-sense AGAGGGAAGAGG GAGAGCTTCTG [SEQ ID NO: 6]. For the PCRreaction, the following were used: 1.5 U/25 μl Taq-Polymerase(Boehringer, Mannheim) and reaction buffer according to themanufacturer's instructions: 200 μM dNTP's; 0.2 μmol each of sense andα-sense primer, 0.5 μl cDNA or genomic DNA from tumour biopsies and celllines; the following reaction steps were performed: 35 cycles, 95° C. 45secs, 72° C. 45 secs.

Analysis by restriction digestion. The transmembrane domain of FGFR-4from genomic or cDNA was amplified as described above. To test biopsiesand cell lines for the G1217A mutation by restriction digestion, the PCRproducts were incubated for 1 hr at 60° C. with 5 U/25 μl of BstN1 (NEB,Schwalbach/Taunus). The DNA fragments from the restriction digestionwere separated with a 20% polyacrylamide gel and stained with ethidiumbromide. The analysis of the wild type receptor yields a 109, a 37 and a22 base-pair sized fragment (track 4). On the other hand, as a result ofthe mutation G1217A a further restriction cleavage site for BstN1 isformed. The mutated receptor shows further 80 and 29 base-pair sizedfragments, while the 109 base-pair sized fragment disappears (track 1:homozygotic; tracks 2 and 3: heterozygotic) (see FIG. 2).

Genotype Analysis of Genomic DNA by Restriction Digestion.

Genomic DNA from the tissue samples of the primary tumours was isolatedby standard methods (Current Protocols in Molecular Biology, John Wileyand Sons, Inc., 1995). In order to be able to genotype analyse thegenomic DNA, the transmembrane region in the FGFR-4 gene was amplifiedwith the following primers in a PCR reaction:

5′-GACCGCAGCAGCGCCCGAGGCCAG-3′ (bp 1129-1142; [SEQ ID NO: 5]), and5′-AGAGGGAAGAGGGAGAGCTTCTG-3′ (bp 1275-1297; [SEQ ID NO: 6]). For thePCR reaction, Ready-to-Go PCR Beats (Pharmacia, Uppsala, Sweden) wereused. The following PCR cycles were used: 3 min at 95° C., 45 secs at94° C., 45 secs at 72° C. and 5 mins at 72° C. A total of 35 cycles wereperformed. The PCR products were incubated for 1 hr at 60° C. with 5U/25 μl of BstN1 (NEB, Schwalbach/Taunus). The DNA fragments from therestriction digestion were separated with a 20% polyacrylamide gel andstained with ethidium bromide. The ³⁸⁸Arg allele is characterised by twofragments of 80 and 29 by size, while the ³⁸⁸Gly allele is indicated bya single 109 by sized fragment. Each genotype analysis was repeatedthree times.

DNA sequencing of PCR products. For the sequence analysis of thetransmembrane domain of FGFR-4, the PCR products were cloned into theBluescript vector. For this, a PCR reaction was performed as alreadydescribed. The following primers were used: sense-GGGAATTCGACCGCAGCAGCGCCCGAGG [SEQ ID NO: 7]; α-sense-GCTCTAGAAGAGGGAAGAGGGAGAG [SEQ ID NO: 8]. The PCR products of the cloning ofFGFR-4^(Arg388)/wt could be directly sequenced in the vector pcDNA3. TheDNA sequencing of plasmid DNA was performed by the chain terminationmethod. After annealing of the T/-primer onto the plasmid DNA, thesequencing reaction was performed with T/-DNA polymerase (Pharmacia,Freiburg). The products of the sequencing reaction were then separatedon a denaturing 5% polyacrylamide gel (7.5 M urea; 1×TBE) and exposed onXray film after drying (see FIG. 3). From this, the DNA sequences of thewild type and also of the mutation, were obtained.

Immunoprecipitation and Western blot analysis. 2.2×10⁶ cells were spreadonto 10 cm Petri dishes and incubated overnight. Then the cell mediumwas replaced by medium with no foetal bovine serum and incubated for afurther 24 hrs. For the stimulation, the cells were incubated for 10mins with 50 ng aFGF/ml, washed twice with cold PBS and placed on ice.The cells were incubated for 15 mins at 4° C. with 300 μl of cold lysisbuffer (1% w/w NP-40, 1.25% w/v sodium deoxycholate, 0.1% w/v SDS, 0.15M NaCl, 0.01 M sodium phosphate, pH 7.2, 2 mM EDTA, 10 mM sodiumfluoride, 1 mM PMSF, 2 μg/ml aprotinin, 1 mM orthovanadate, 10 mM sodiumpyrophosphate), and the lysate clarified by centrifugation (13,000 RPM)at 4° C. For the protein value determination, the Micro-BCA ProteinAssay (Pierce) was used in accordance with the manufacturer'sinstructions. For the immuno-precipitation, the cell lysates wereadjusted to equal protein content and then incubated for

18 hrs at 4° C. with 0.5 μg anti-FGFR-4 (Santa Cruz) andprotein-A-Sepharose (Pharmacia Freiburg) on a rotating wheel. The immunecomplexes were washed 4 times with cold HNTG (20 mM HEPES pH 7.5, 150 mMNaCl, 0.1% Triton X100, 10% glycerine, 10 mM sodium pyrophosphate). Forsample preparation, the immune complexes were treated with

50 μl 3×Laemmli buffer and incubated for 5 mins at 99° C. Theprecipitated proteins were separated on a 7.5% SDS-PAGE (see FIG. 1).

For Western blots, the proteins separated by SDS-PAGE were transferredto nitrocellulose. Non-specific protein binding sites on the membranewere blocked by incubation for 2 hrs at room temperature withTBS-T/0.25% gelatine (10 mM Tris/HCl pH 8.0, 0.15 M NaCl, 0.05%Tween20). The incubation with primary antibodies was effected for 6 hrsat 4° C. on a tilt shaker. Non-specifically bound antibodies were thenremoved by washing 4 times with

TBS-T/0.25% gelatine. The binding of secondary antibodies was effectedfor 1 hr at room temperature. The non-specifically bound secondaryantibodies were removed by a further washing step. Immune complexes weremade visible with the ECL™ kit (Amersham, Braunschweig) in accordancewith the manufacturer's instructions.

Statistical Methods. Statistical calculations were performed with theaid of the statistics program MedCalc (MedCalc Software, Belgium) andEpiInfo 6.04b (CDC, Atlanta, Ga.). In order to determine thecorrelations between the genotypes in the different patient groups andthe clinical data, an odds ratio, the confidence interval (CI) and astatistical significance (P value) were calculated. Because of the smallnumber of ³⁸⁸Arg homozygotic patients, this group was combined with thegroup of ³⁸⁸Arg heterozygotic patients for the statistical calculations.

Detection of FGFR-4 in Tumour Cell Lines. Table 1 shows the correlationbetween the expression of RTK and breast cancer. Expression of RTKclearly occurs more often in cell lines from breast cancer, while noexpression is detectable in cell lines of normal breast epithelialcells.

TABLE 1 Detection of FGFR-4 in Breast Cancer Cells Northern Blot [sic]FGFR-4 Breast Cancer Cell Lines  1 HTB-30 (SK-BR-3) ++  2 HTB-122(BT-549) −  3 MCF-7 +  4 BT-483 +++  5 T-47D +  6 ZR-75-1 +++  7MDA-MB-468 −  8 MDA-MB-453 ++++  9 MDA-MB-361 ++++ 10 MDA-MB-415 − 11MDA-MB-231 − Normal Breast Epithelial Cell Lines 12 HBL-100 − 13 MCF-10A− Key: −: no expression +: expression ++: strong expression +++: verystrong expression ++++: extreme expression.

From Table 2, it is clear that the G388R mutation also occurs in celllines of other cancer types and is correlatable with these. In healthyepithelial cell lines, the mutation is not detectable.

TABLE 2 Mutation FGFR-4 G388R in various other tumour cell lines Samplegenomic DNA cDNA Glioblastoma U-138 −/− −/− U-373 −/− −/− U-172 −/− −/−U-118 −/* −/* SF-763 −/− −/− U-1240 */* */* T-98G (*)/− (*)/− U-937 −/−−/− Neuroblastoma SK-N-SH −/* −/* SH-SY-SY −/* −/* Uterine Cancer OAW-42−/* −/* PA-1 −/− −/− Caov-3 −/− −/− Squamous Hlac-78 −/− −/− Hlac-79 −/−−/− Scc-4 −/− −/− Scc-10a −/− −/− Scc-10b −/− −/− Scc-17a −/− −/−Scc-17b −/− −/− Scc-22a */* */* Scc-22b */* */* HaCat −/− −/− FaDu −/−−/− Normal Breast Epithelial Cell Lines HBL-100 −/− −/− MCF-10A −/− −/−Key: −/− homozygotically nonmutated */− heterozygotically mutated */*homozygotically mutated

Detection of the FGFR-4 Mutation G388R in Biopsies. Table 3 thus showsthat of 61 female patients from St Petersburg with breast cancer whowere studied, 56% carried the G388R mutation, 45% of themheterozygotically and 11% homozygotically. Of the 69 female breastcancer patients from Munich who were studied, 43% carried the G388Rmutation, 32% of them heterozygotically and 11% homozygotically. Theproportion of the total percentage of the mutation in female patientsfrom St Petersburg and Munich is different. This suggests that the G388Rmutation is a germ line mutation.

TABLE 3 Detection of FGFR-4 mutation G388R in biopsies Samples frombreast tumours From St Petersburg From Munich Sample gen. DNA Samplegen. DNA cDNA 19 102 T */− 5382 T */− */− 20 102 N 5609T */* */* 21 103T */− 8926 T */− */− 22 103 N */− 9456 T */* */* 23 2 T */* 9556 T */−*/− 24 2 N */* 10347 T −/− −/− 25 12 T −/− 10555 T −/− −/− 26 12 N 10681T */− */− 27 13 T −/− 10781 T */− */− 28 13 N 10808 T */* */* 29 14 T*/− 11189 T */− */− 30 14 N */− 11526 T */− */− 31 15 T −/− 11697 T */−32 15 N 11820 T −/− −/− 33 17 T */− 12015 T −/− −/− 34 17 N */− 12166 T*/− */− 35 18 T −/− 13932 T */− */− 36 18 N 16003 T */− */− 37 20 T −/−16353 T −/− −/− 38 20 N 1 N 39 21 T */* 2 T */− */− 40 21 N */* 3 N 4122 T */− 4 T −/− −/− 42 22 N 5 N 43 23 T */− 6 T −/− −/− 44 23 N 7 N 4531 T */− 8 T −/− −/− 46 31 N */− 9 N 47 42 T −/− 10 T */− */− 48 42 N 11N 49 43 T −/− 12 T −/− −/− 50 43 N 13 N 51 45 T −/− 14 T */− */− 52 45 N16 T */− */− 53 47 T */− 17 N −/− 54 47 N */− 18 T */− 55 48 T −/− 19 N56 48 N 20 T −/− 57 50 T −/− 38 T −/− 58 50 N 3433 T −/− 59 53 T −/−3539 T 60 53 N 3631 T */* 61 54 T */− 3632 T −/− 62 54 N */− 3636 T −/−63 55 T −/− 3637 T */− 64 55 N 3638 T −/− 65 60 T */− 3640 T −/− 66 60 N*/− 991 N −/− 67 61 T */* 991 T −/− 68 61 N */* 15153 N 69 62 T */−15153 T −/− 70 62 N */− 15856 N */* 71 63 T */− 15856 T */* 72 63 N */−12845 N 73 67 T */− 12845 T −/− 74 67 N */− 19044/93 N 75 69 T −/−19044/93 T −/− 76 69 N 9426/93 N 77 78 T */− 9426/93 T −/− 78 78 N −/−2005 N */− 79 79 T */* 2005 T */− 80 79 N */* 14860 N 81 82 T −/− 14860T −/− 82 82 N 4198 T −/− 83 83 T −/− 5739 T */* 84 83 N 6060/93 tum */*85 85 T −/− 6982/93 tum −/− 86 85 N 7244/93 tum −/− 87 86 T */− 8114/93tum −/− 88 86 N */− 8335/93 tum */− 89 87 T −/− 8481/93 tum */− 90 87 N8566/93 tum −/− 91 89 T −/− 8786/93 tum */* 92 89 N 9145/93 tum −/− 9394 T −/− 9354/93 tum −/− 94 94 N 9796/93 tum −/− 95 97 T */* 9798/93 tum−/− 96 97 N */− 10125/93 tum −/− 97 98 T −/− 10150/93 tum */− 98 98 N11218/93 tum −/− 99 99 T */* 11673/93 tum −/− 100 99 N */* 13232/93 tum−/− 101 100 T −/− 13316/93 tum */− 102 100 N 14724/93 tum −/− 103 101 T*/− 14879/93 tum #1 −/− 104 101 N */− 14879/93 tum #2 −/− 105 102 T(#20) */− 15645/93 tum −/− 106 102 N (#19) */* 107 103 T (#22) */− 108103 N (#21) 109 104 T */− 110 92 T */* 111 65 T 112 52 T */− 113 35 T*/− 114 33 “A” T */− 115 33 “B” mts. */− 116 30 T */− 134 30 N */− 11727 T */* 133 27 N */* 118 24 T */− 119 10 T */− 132 10 T */− 120 3 T −/−121 90 T −/− 122 90 N 123 80 T −/− 124 80 N 125 81 T −/− 126 58 T −/−127 51 T */− 128 51 N 129 44 T */− 130 44 N

Correlation between the FGFR-4-G388R mutation and the detection ofFGFR-4 expression. From Table 4 below, it is clear that the G388Rmutation (genomic DNA and cDNA) only occurs when expression and/orintensified expression occurs. The mutation is detectable neither in thenormal breast epithelial cell lines nor in the breast cancer cell linesin which no RTK expression was found.

The cell line MDA-MB 453, whose RTK expression is especially pronounced,shows a homozygotic G388R mutation.

TABLE 4 Correlation between the FGFR-4-G388R mutation and the detectionof FGFR-4 expression Northern Blot Mutation FGFR-4 cDNA gen. DNA Breastcancer cell line  1 HTB-30 (SK-BR-3) ++ +/+ +/+  2 HTB-122 (BT-549) −−/− −/−  3 MCF-7 + +/− +/−  4 BT-483 +++ +/− +/−  5 T-47D + +/− +/−  6ZR-75-1 +++ +/− +/−  7 MDA-MB-468 − −/− −/−  8 MDA-MB-453 ++++ +/+ +/+ 9 MDA-MB-361 ++++ +/− +/− 10 MDA-MB-415 − −/− −/− 11 MDA-MB-231 − +/−+/− Normal Breast Epithelial Cell Lines 12 HBL-100 − −/− −/− 13 MCF-10A− −/− −/− Key: −: no expression +: expression ++: strong expression +++:very strong expression ++++: extreme expression. −/−: no mutation */−:heterozygotically mutated */*: homozygotically mutated

Study of the Correlation Between the Occurrence of the FGFR-4 MutationG388R and Lymph Node Metastasis Status or Relapse-Free Survival Time

Table 5 shows the clinical parameters of all patients who took part inthe study of the role of the G388R mutation in the tumorigenesis ofbreast cancer. It is found that patients with a G388R mutation have aworse long-term prognosis than patients with no G388R mutation.

Key to Table 5;[on following pages]

Her2: expression level of the Her2 receptor; 0=no expression to3=overexpression

OPDAT: date of the operation

M/R: metastasis formation/relapse; 0=no /1=yes

Vers: died; 0=no/1=yes

ÜBERRE: survival time without relapse, in months

Grade: differentiation grade of tumour; 1=strong differentiation/3=lowdifferentiation

stage: size of the primary tumour.

E-Rec: expression of the oestrogen receptor; 0=no expression to12=highest expression

GEN: genotype of the FGFR-4; G=wild type allele; R=mutated allele

BEDAT: date of last observation

REZDAT: date of relapse diagnosis

TODDAT: date of death

ÜBERLEB: survival time overall

Nod.: metastases in the lymph nodes; 0=no/1=yes

Men: menopause

P-Rec: expression of the progesterone receptor: 0=no expression to12=highest expression

TABLE 5 FGFR-4 genotypes and clinical case data PathoNr. Her2 GEN OPDATBEDAT M/ REZDA Ver TODDA ÜBERR Überle Alter stag Nod. Grad Men E- P-489292 G/G 31.03.92 14.02.97 0 0 59 59 71.7 1c 0 2 2 12 9 617792 G/G24.04.92 14.07.94 0 0 27 27 1 639792 0 G/G 29.04.92 03.04.94 0 0 23 23 0724493 G/G 11.05.93 23.06.97 0 0 48 48 64.3 2 1 2 2 12 12 914593 0 G/G16.06.93 07.03.96 0 0 33 33 86.2 2 2 2 8 9 935493 2+ G/G 21.06.9315.03.96 0 0 33 33 49.6 2 1 3 2 0 0 963392 G/G 30.06.92 30.06.97 0 0 6060 79.4 1b 0 2 2 3 12 979693 G/G 29.06.93 07.03.96 0 0 33 33 46.7 2 0 23 9 0 979893 1+ G/G 29.06.93 30.06.97 0 0 48 48 77 4b 1 2 2 1 0 10347920 G/G 13/07.92 26.03.97 0 0 56 56 43.5 3 1 2 1 0 6 1323293 G/G 02:03.9305.08.97 0 0 48 48 61.4 2 1 3 2 0 0 1331693 3+ G/G 30.09.92 29.03.95 0 036 36 71.2 2 1 2 2 6 1 1564593 1+ G/G 19.10.92 19.06.97 0 0 56 56 65.3 21 2 2 0 0 78692 G/G 19.01.88 29.06.93 0 0 65 65 63.8 2 0 3 2 4 12 1769893+ G/G 08.02.85 06.02.92 0 0 84 84 43.4 2 1 2 1 273690 1+ G/G 22.02.8614.02.92 0 0 72 72 52.3 2 0 2 1 12 12 725289 G/G 14.12.87 0 49.5 2 0 2 00 729991 0 G/G 23.05.87 22.02.92 0 0 57 57 65.7 2 1 2 2 12 12 733290 1+G/G 28.05.86 28.03.87 0 1 28.03.87 14 14 81.7 1b 0 2 2 12 12 826790 2+G/G 19.06.86 22.01.94 0 0 91 91 77.7 2 0 2 2 9 0 867191 0 G/G 19.06.8722.01.94 0 0 79 79 77.5 2 0 3 2 0 0 988590 G/G 24.07.86 22.01.94 0 0 9090 61.5 1b 0 2 2 6 9 991790 0 G/G 23.07.86 25.03.92 0 0 68 68 50.7 1c 02 2 8 4 1031190 2+ G/G 30.07.86 26.01.94 0 0 90 90 43.4 1c 0 2 1 10333911+ G/G 21.07.87 03.03.92 0 0 55 55 62.4 2 1 3 2 12 12 1055592 G/G14.07.88 25.04.93 0 1 25.04.93 57 79 77.8 2 0 2 2 6 12 1101191 0 G/G31.07.87 03.06.93 0 0 70 70 48.5 2 0 2 1 0 0 1426491 1+ G/G 08.10.8704.02.94 0 0 76 76 54.9 1b 0 3 2 0 0 1560492 G/G 22.09.88 02.04.92 0 042 42 50 1c 0 3 3 0 0 1605790 0 G/G 25.11.86 20.02.92 0 0 63 63 56.4 1c1 2 2 12 6 1641488 2+ G/G 21.12.84 04.02.94 0 0 109 109 67.4 1b 0 2 2 91121893 1+ G/G 23.07.93 18.01.96 1 06.09.94 1 18.01.96 14 16 59.9 2 1 32 4 2 1201592 1+ G/G 10.08.92 25.04.95 1 25.04.95 0 29 29 48.9 1c 1 2 11 6 258093 0 G/G 22.02.85 18.04.91 1 30.11.89 1 18.04.91 57 102 79.4 1c1 2 2 479090 3+ G/G 04.04.86 23.07.89 1 12.04.88 1 23.07.89 24 55 67.91c 0 2 2 0 0 806490 0 G/G 14.06.86 18.09.90 1 10.11.88 1 18.09.90 29 7166.7 2 1 2 2 8 0 963589 1+ G/G 26.07.85 06.03.89 1 26.04.88 1 06.03.8933 60 47.8 2 1 3 1 0 972291 3+ G/G 09.07.87 19.08.89 1 16.07.88 119.08.89 12 35 48.3 2 0 3 1 0 0 995589 0 G/G 01.08.85 27.06.88 129.04.87 1 27.06.88 21 48 58.4 3 1 3 2 6 1112389 3+ G/G 30.08.8514.07.86 1 14.06.86 1 14.07.86 9 14 79.4 4b 2 2 2 0 0 1726892 G/G18.11.88 23.04.91 1 07.11.90 1 23.04.91 24 40 38.7 2 0 3 1 0 0 289791 3+G/R 25.02.87 02.04.92 0 0 61 61 48.1 2 1 3 2 0 0 337293 G/R 03.03.8904.03.92 0 0 36 36 51.2 1c 0 2 1 6 2 879290 2+ G/R 01.07.86 01.10.92 0 101.10.92 75 103 44.9 1c 0 3 1 1 6 893090 0 G/R 03.07.86 17.12.93 0 0 8989 68.5 4 0 3 2 12 6 1106192 G/R 23.07.88 04.02.94 0 0 66 66 53.9 1c 0 22 2 12 1107789 G/R 29.08.85 13.02.92 0 0 77 77 50.3 1c 0 2 1 8 121113892 G/R 24.07.92 14.10.97 0 0 63 63 51.9 2 0 1 2 2 6 1118990 G/R19.08.86 15.02.92 0 0 66 66 53.8 1c 0 2 2 6 4 1152692 3+ G/R 31.07.9230.06.97 0 0 59 59 57.7 2 1 2 2 4 12 1599789 1+ G/R 14.12.85 15.02.87 01 15.02.87 14 19 80.2 1c 0 3 2 3 2 1614591 0 G/R 12.11.87 25.02.92 0 051 51 76.7 2 1 2 2 3 9 92390 0 G/R 20.01.86 13.02.92 1 09.02.91 0 61 7345.8 2 1 3 1 0 1 99289 0 G/R 23.01.85 17.03.92 1 22.02.90 0 61 86 47.11c 1 2 1 130588 2+ G/R 20.01.84 30.01.92 1 18.02.87 0 37 96 39 2 1 2 1306490 3+ G/R 01.03.86 06.06.87 1 06.11.86 1 06.06.87 8 21 52.2 2 1 3 30 0 492191 3+ G/R 07.04.87 20.04.94 1 10.10.90 0 42 84 56.2 2 1 3 2 3 0529692 G/R 07.04.92 29.03.96 1 23.02.95 1 29.03.96 34 46 66.9 1b 0 3 2 00 529990 3+ G/R 17.04.86 29.04.87 1 14.04.87 1 29.04.87 12 17 77.1 2 1 32 0 0 538292 3+ G/R 07.04.92 13.10.93 1 07.04.92 1 13.10.93 0 18 56.1 42 3 2 0 0 614091 2+ G/R 29.04.87 21.04.90 1 31.07.88 1 21.04.90 15 4947.2 2 2 2 1 2 6 651591 0 G/R 07.05.87 17.09.93 1 14.04.90 0 35 76 49.33 1 2 1 3 9 673592 G/R 06.05.92 27.02.96 1 06.06.93 0 13 45 36.8 2 0 2 10 0 678092 2+ G/R 07.05.92 29.06.96 1 07.05.92 0 0 49 56.7 1c 1 2 2 0 0714289 2+ G/R 04.06.85 16.09.89 1 30.06.87 1 16.09.89 25 71 55 1c 1 2 28 807492 0 G/R 29.05.92 29.11.96 1 29.11.96 0 54 54 1 848193 G/R03.06.93 10.09.94 1 16.08.94 1 10.09.94 15 16 58 2 2 2 2 0 4 955692 1+G/R 29.06.92 26.11.96 1 17.08.95 37 53 49.9 1c 1 3 3 2 1 1022090 0 G/R29.07.86 02.07.88 1 23.06.88 1 02.07.88 23 32 51.7 2 2 2 2 12 1 1054987G/R 05.08.83 15.12.84 1 29.02.84 1 15.12.84 7 23 71.5 2 0 3 2 1078192 2+G/R 20.07.92 22.10.95 1 25.09.95 0 38 39 79.4 2 1 3 2 1 9 1079689 2+ G/R22.08.85 27.03.91 1 31.12.89 1 27.03.91 52 93 81.4 4 2 3 2 1169792 2+G/R 04.08.92 01.07.95 1 01.07.95 1 01.07.95 47 47 67.3 2 1 3 2 6 91216692 0 G/R 12.08.92 30.07.96 1 09.09.94 0 23 48 45.3 2 1 2 1 4 61314689 2+ G/R 16.10.85 21.02.92 1 05.11.91 0 73 76 81.2 4 1 2 2 6 121391992 2+ G/R 17.09.92 01.05.93 1 15.01.93 1 01.05.93 5 8 76 2 1 3 21696290 G/R 11.12.86 20.02.92 1 30.11.89 0 36 62 55.2 2 0 2 2 6 6 920891G/R 50.5 2 0 3 1 2 6 213593 R/R 10.05.89 20.04.94 0 0 62 62 45.3 1c 0 21 3 6 313791 3+ R/R 28.02.87 21.02.92 0 0 60 60 68.6 2 1 3 2 0 0 878693R/R 09.06.93 07.09.96 0 0 33 33 0 1107391 3+ R/R 01.08.87 29.02.92 0 055 55 61.5 1c 0 2 2 3 1 1125690 R/R 20.08.86 13.10.93 0 0 86 86 43.1 3 13 1 0 0 120788 R/R 27.01.84 24.04.86 1 28.01.86 1 24.04.86 24 37 68.1 21 3 2 560992 3+ R/R 13.04.92 04.04.93 1 10.04.92 1  4.4.93 0 12 59.1 4b2 3 2 6 0 1008692 2+ R/R 08.07.92 11.07.94 1 17.08.93 1 11.07.94 13 2447.4 2 1 3 2 0 0 1686490 3+ R/R 10.12.86 23.07.87 1 07.05.87 1 23.07.875 10 55.4 2 1 3 2 0 0

From FIG. 4, it is clear that the G388R mutation is to be found ingreater number in patients who already have metastases in the lymphnodes at the time of the first treatment. Of the patients with a G388Rmutation, 62.7% had lymph node metastases, while of the patients with noG388R mutation only 38.2% displayed metastases in the lymph nodes. Asthe lymph node metastasis status is an important prognostic marker forthe further discrimination of tumours with a worse and those with abetter prognosis, it can be concluded from this result that the G388Rmutation in the 85 patients studied leads to a more severe tumourprogression.

From FIG. 5 it is can be seen that in the group of patients studied, therelapse-free survival probability is very much lower for those with aG388R mutation than for those patients who have no G388R mutation. While74.4% of the patients with a relapse possess the 388R genotype, only25.6% have the 388G genotype. This shows that patients with the G388Rmutation suffer a relapse more quickly, and therefore could not besuccessfully treated.

In summary, it can be stated that the FGFR-4 mutation G388R leads to a2.7-fold (OR=2.7; CI: 1.02<OR<7.4) increased risk of metastasisformation in the lymph nodes and to a 5.44-fold (OR=5.44; CI:1.93<OR<7.4) increased risk of a tumour relapse. Patients with a mutatedFGFR-4 allele (G388R) thus seem to have a predisposition to a tumourrelapse and hence a poorer disease prognosis.

Materials Acrylamide Serva, Heidelberg Agar Difco, Detroit Agarose BRL,Eggenstein Ampicillin Boehringer, Mannheim Aprotinin Sigma, TaufkirchenN,N′-bisacrylamide Roth, Karlsruhe Caesium chloride BRL, EggensteinDesoxynucleotides Pharmacia, Freiburg Ethidium bromide Sigma,Taufkirchen Gelatine Sigma, Taufkirchen Guanidium isothiocyanate Fluka,Switzerland HEPES Serva, Heidelberg Sodium fluoride Sigma, TaufkirchenPMSF Sigma, Taufkirchen SDS Roth, Karlsruhe Tris Riedel de Haen, SeelzeTriton X100 Serva, Heidelberg Tween20 Sigma, Taufkirchen

All substances not listed here came from the firms Sigma (Taufkirchen),Serva (Heidelberg), Riedel De Haen or Merck (Darmstadt) and the highestpossible purity grades were used.

Instruments Electrophoresis of DNA Workshop, MPI for Biochemistry,Electrophoresis of proteins Martinsried Atto, Japan Refrigeratedcentrifuge Biofuge 17S Heraeus, Hanau Protein transfer Semidry blotapparatus, Workshop, MPI for Biochemistry, Martinsried Sterile workbenchBiogard, The Baker Company, USA Cell culture Incubator B5060 EK/CO₂,Heraeus, Hanau Cell counting Coulter Counter, Coulter Electronics,Glasgow.

LITERATURE

-   Basilico C and Moscatelli D: The FGF family of growth factors and    oncogenes. Advanc. Cancer Res. 59, 115-164 (1992).-   Coulier F, De Lapeyriere 0 and Birnbaum D: Complexity of the FGF    family: the proof by 9. Méd./Sci. 9, 1113-1115 (1993).-   Folkmann J and Klagsbrun M: Science 235, 442-447 (1987).-   Gival D and Yayon A: Complexity of FGF receptors: genetic basis for    structural diversity and functional specificity, FASEB J. 6:    3362-3369 (1992).-   Johnson D E and Williams L T: Structural and functional diversity in    the FGF receptor multigene family. Adv. Cancer Res. 60:1-41 (1993).-   Kimelman D, Abraham J A, Haaparanta T, Palisi T M and Kirschner M W:    Science, 242, 1053-1058 (1988).-   Mohammadi M et al., Science 776, 955-959 (1997).-   Slack J M W, Darlington G G, Heath J K and Godsave S F: Nature 326,    197-200 (1987).-   Thomas K A, Rios-Candelore M, Giminez-Gallego G, DiSalvo J, Bennett    C, Rodkey J and Fitzpatrick S: Proc. Natl. Acad. Sci. USA, 82,    6409-6413 (1985).-   Thompson J A, Haudenschild C C, Anderson K D, DiPietro J M, Anderson    W F and Maciag T. (1989): Proc. Natl. Acad. Sci. USA, 86, 7928-7932    (1989).

Cell Line Origin No. U-138 ATTC HTB-16 U-373 ATTC HTB-17 U-172 U-118ATTC HTB-15 SF-763 SUGEN U-1240 SUGEN T-98G SUGEN U-937 ATCC CRL-1593SK-N-SH ATCC HTB-11 SH-SY5Y F. J. Klinz OAW-42 DKFZ PA-1 ATCC CRL-1572Caov-3 ATCC HTB-75 Hlac-78 Dr. Wustrow Hlac-79 Dr. Wustrow Scc-4 ATCCCRL-1624 Scc10a Dr. Wustrow Scc10b Dr. Wustrow Scc22a Dr. Wustrow Scc22bDr. Wustrow Scc17a Dr. Wustrow Scc17b Dr. Wustrow FaDu Dr. Wustrow HaCatHBL100 ATCC HTB-124 MCF10A ATCC CRL-10317 SKBr-3 ATCC HTB-30 BT-549 ATCCHTB-122 MCF-7 ATCC HTB-22 BT483 ATCC HTB-121 T-47-D ATCC HTB-133 ZR-75-1ATCC CRL-1500 MDA-MB-468 ATCC HTB-132 MDA-MB-453 ATCC HTB-131 MDA-MB-361ATCC HTB-27 MDA-MB-415 ATCC HTB-128 MDA-MB-231 ATCC HTB-26 K-562 ATCCCCL-243

1-32. (canceled)
 33. A method for the prophylactic and/or therapeutictreatment of a receptor tyrosine kinase (RTK)-hyperfunction-induceddisorder in a mammal, which method comprises administering to a mammalan effective amount of at least one inhibitor of fibroblast growthfactor receptor-4 (FGFR-4), wherein the RTK-hyperfunction-induceddisorder is treated prophylactically and/or therapeutically.
 34. Themethod of claim 33, wherein the RTK-hyperfunction-induced disorder isone or more disorders selected from the group consisting of cancer, adisease attributable to cellular hyperproliferation and/or cellularinvasion of tissue, a carcinoma, and a metastasis.
 35. The method ofclaim 34, wherein the disorder is breast cancer, squamous cellcarcinoma, glioblastoma, neuroblastoma, or uterine cancer.
 36. Themethod of claim 33, wherein the inhibitor is a kinase-inactive receptor.37. The method of claim 33, wherein an overexpression and/or an alteredactivity of FGFR-4 is lowered and/or inhibited.
 38. The method of claim37, wherein the overexpression and/or the altered activity of FGFR-4 istriggered by a mutation of the FGFR-4.
 39. The method of claim 38,wherein the mutation is one or several point mutations.
 40. The methodof claim 39, wherein the one or several point mutations leads to anexchange of a hydrophobic amino acid for a hydrophilic amino acid. 41.The method of claim 38, wherein the mutation occurs in the transmembranedomain of FGFR-4.
 42. The method of claim 41, wherein the mutation isone or several point mutations that lead to an exchange of a hydrophobicamino acid for a hydrophilic amino acid.
 43. The method of claim 38,wherein the mutation occurs at amino acid position 388 in the FGFR-4molecule.
 44. The method of claim 43, wherein the mutation leads to anexchange of glycine for arginine.
 45. The method of claim 38, whereinthe mutation is a germ line mutation.
 46. The method of claim 33,wherein the FGFR-4 is mutated and the inhibitor inhibits the mutatedFGFR-4.
 47. A mutated FGFR-4 which is overexpressed and/or has alteredactivity in a cell.
 48. The mutated FGFR-4 of claim 47, wherein ahydrophobic amino acid in the wild-type FGFR-4 has been exchanged for ahydrophilic amino acid in the mutated FGRR-4.
 49. The mutated FGFR-4 ofclaim 47, which comprises a point mutation in the transmembrane domain.50. The mutated FGFR-4 of claim 49, wherein the point mutation occurs atamino acid 388 and, optionally, results in replacement of glycine witharginine.
 51. An isolated DNA or RNA molecule encoding the mutatedFGFR-4 of claim
 47. 52. An isolated DNA or RNA molecule encoding themutated FGFR-4 of claim
 48. 53. An isolated DNA or RNA molecule encodingthe mutated FGFR-4 of claim
 49. 54. An isolated DNA or RNA moleculeencoding the mutated FGFR-4 of claim
 50. 55. A method of diagnosing anRTK-hyperfunction-induced disorder or a genetic predisposition thereforin a mammal, which method comprises determining the presence of amutated FGFR-4 protein or a nucleic acid encoding a mutated FGFR-4protein in a sample of protein or nucleic acid, respectively, obtainedfrom the mammal, wherein the presence of such a protein or nucleic acidis indicative of an RTK-hyperfunction-induced disorder or a geneticpredisposition therefor.
 56. The method of claim 55, wherein theRTK-hyperfunction-induced disorder is cancer.
 57. The method of claim55, which method comprises contacting the sample of nucleic acid with alabeled DNA or RNA molecule encoding a mutated FGFR-4 under hybridizingconditions and detecting the labeled DNA or RNA molecule afterhybridization, wherein the detection of the labeled DNA or RNA isindicative of the presence of a nucleic acid molecule encoding a mutatedFGFR-4 in the sample.
 58. The method of claim 55, which method comprisescontacting the sample of nucleic acid with a restriction enzyme whoserecognition sequence is affected by the mutation in the mutated FGFR-4and detecting the presence or absence of fragments or the presence ofaltered fragments of the nucleic acid after contact with the restrictionenzyme, wherein the absence of fragments or the presence of alteredfragments of the nucleic acid after contact with the restriction enzymeis indicative of the presence of a nucleic acid molecule encoding amutated FGFR-4 in the sample.
 59. The method of claim 58, wherein themutation in the mutated FGFR-4 occurs at amino acid position
 388. 60. Amethod of identifying an inhibitor of tyrosine kinase activity, whichmethod comprises contacting a potential inhibitor with a mutated FGFR-4and determining tyrosine kinase activity in the absence and presence ofthe potential inhibitor, wherein a decrease in tyrosine kinase activityin the presence of the potential inhibitor indicates that the potentialinhibitor is an inhibitor of tyrosine kinase activity.
 61. A method oftreating cancer in a mammal, which method comprises administering to themammal an effective amount of a kinase-inactive receptor or an inhibitorthat inhibits FGFR-4, whereupon the cancer is treated.
 62. An antibodythat reacts specifically with the mutated FGFR-4 of claim
 47. 63. Anantibody that reacts specifically with the mutated FGFR-4 of claim 48.64. An antibody that reacts specifically with the mutated FGFR-4 ofclaim
 49. 65. An antibody that reacts specifically with the mutatedFGFR-4 of claim 50.